1. Field of Invention
This invention relates to therapeutic compounds that improve the outcome of preterm deliveries. More specifically, this invention relates to the usage of compounds that are free radical scavengers or that promote the production of free radical scavengers to prevent adverse fetal outcomes in conditions with high free radical production, preterm deliveries or premature rupture of membranes or even inhibit preterm deliveries.
2. Description of Related Art
Clinical Context
Preterm birth is the major cause of perinatal morbidity and mortality in the world.
Prematurity is responsible for 75% of infant mortality and 50% of long-term neurological handicaps, including blindness, deafness, developmental delay, cerebral palsy, and chronic lung disease (Berkovitz and Papiemik, 1993; Creasy 1993). Any treatment that alters the events of preterm birth or prevents it could profoundly reduce neonatal mortality and morbidity. Even a relatively brief delay in the timing of delivery can have major benefit. Survival rates improve by some 2% per day from the 23rd to the 26th week of pregnancy (i.e. from 16% at 23 weeks to 57% at 26 weeks) reaching 80% at 28 weeks and over 90% by 30 weeks of gestation (Haywood et al., 1994). Yet, there are no agents that prolong pregnancy complicated by preterm labor by more than 48 hours compared to placebo. The health care costs from prematurity are enormous. It is estimated that the total cost per survivor with a birthweight of less than or equal to 900 g (approximately 27 of 40 weeks) in the U.S. exceeds his or her's total average life-time earnings. Over 4 billion dollars (35% of health care costs for all infants) is spent for the care of low-birth-weight infants (Iams, 1995).
The cause of most preterm births is unknown (Bernstein et al., 1998). Current evidence suggests the etiology is multifactorial with a fetal inflammatory syndrome contributing to a large proportion. Both intrauterine (chorioamnionitis) and systemic infections are proposed as important causes of preterm labor (Romero et al., 1990). Systemic maternal infections such as pyelonephritis, pneumonia, syphilis and malaria, for example, are all associated with preterm labor and preterm birth (Gibbs et al., 1992). Colonization of the lower genital tract with a variety of microorganisms may lead to ascending intrauterine infection that in turn results in preterm labor. Microbial invasion of the amniotic cavity occurs in 10% of the patients with preterm labor and intact membranes (Romero et al., 1990) and in 38% of the patients with preterm premature rupture of membranes (PPROM) (Romero et al., 1990). Molecular biologic techniques such as PCR, which is more sensitive than culture, detect bacteria in 60% of the pregnancies complicated with preterm labor (Markenson et al., 1997). Infection-related preterm labor likely involves the release of inflammatory cytokines host defense mechanisms in response to bacterial products (i.e. lipopolysaccharide: LPS). It is believed that the pro-inflammatory cytokines (IL-1, TNFα, IL-8 etc.) stimulate the production of uterotonins (agents that cause uterine contractions), such as prostaglandins, leukotrienes and oxytocin, by the decidua and fetal membranes, eventually leading to the onset of labor. The cytokines may also trigger local mechanisms of cervical ripening and maturation of fetal membranes, that requires the recruitment of inflammatory cells, the release of metalloproteinases (MMPs), and finally the degradation of the extracellular matrix, leading to effacement and dilatation of the uterine cervix or to the rupture of fetal membranes (Romero et al., 1988).
Recently, it was suggested that the levels of IL-6, a pro-inflammatory cytokine, in the fetal circulation correlate strongly with poor neonatal outcome (Gomez et al., 1998). This correlation suggests that at least some neonatal complications are not caused by prematurity per se but are the effect of a fetal inflammatory syndrome. In other words, the stimulus for preterm birth may also directly or indirectly adversely affect the fetus probably through maternal or fetal adaptive responses that may become maladaptive (such as excessive inflammation). This hypothesis is supported by the observation that the neonatal mortality rate is increased in the presence of chorioamnionitis when adjusted for gestational age at delivery (Seo et al., 1992). Furthermore, neonatal morbidity is also significantly increased in the presence of chorioamnionitis. An increased number of inflammatory cells are described in the lungs of infants born to mothers with chorioamnionitis and the resulting pneumonitis lesion is considered a contributor to the increased chronic lung diseases so highly prevalent in these infants (Grigg et al., 1993; Waterberg et al., 1996). Even long-term handicaps such as cerebral palsy are significantly associated with intrauterine infection when controlled for gestational age (Murphy et al., 1995).
Ultrasonographically detectable neonatal brain white matter lesions are the most important prospectively identifiable risk factor for cerebral palsy, defined as sustained neurologic disability with aberrant control of movement and posture appearing early in life. The presence of these lesions are significantly correlated with the level of inflammatory cytokines in amniotic fluid (Yoon et al., 1997a) and in the brain at post-mortem, the periventricular leukomalacia lesions where examined immunohistochemically for the presence of IL-1β, IL-6 and TNFα (Yoon et al., 1997b). However, antagonists of IL-1β or TNFα where not sufficient in preventing either preterm birth or fetal lethality in an endotoxin-injected mouse model suggesting that these cytokines may be only an associated marker of the mounted inflammatory response (Fidel et al., 1997).
Pathophysiological Significance of the Oxidant/Antioxidant Balance
Oxygen (O2) is paramagnetic in the ground state and contains two unpaired electrons whose spins are parallel. This results in a spin restriction that hinders the insertion of pairs of electrons and favors a univalent reduction (Taube, 1965). The univalent reduction of dioxygen to water involves the formation of partially reduced intermediates, (reactive oxygen species; ROS) such as superoxide radical-anion (O2.−), hydrogen peroxide (H2O2), and the hydroxyl radical (OH.). These partially reduced intermediates are very reactive and are the cause of oxygen toxicity and mutagenicity (Moody et al., 1982). Most living organisms have evolved well-integrated antioxidant defense mechanisms (scavengers) that include superoxide dismutases (SOD), catalase, glutathione peroxidases, reduced glutathione (GSH), β-carotene, and vitamins C (ascorbic acid) and E. Under normal respiration, a small but significant amount (1-5%) of the total oxygen consumed is reduced via the univalent pathway. Thus, ROS are normal products of the biological reduction of oxygen and their steady-state concentration is kept low by the above noted scavengers. There could be, however, another evolutionary reason for ROS formation: signal transduction (Storz et al., 1990). Most sources of ROS involved in signal transduction seem to initially generate O2.−, with hydrogen peroxide (H2O2) being formed as a result of dismutation of O2.−.
a) Modulation of Cytokine Production
Several lines of evidence demonstrate that the inflammatory response (as reflected in the cytokine levels) to bacteria or bacterial products (e.g. LPS) is dependent on the oxidant/antioxidant balance. An increase in pro-oxidants or a decrease in antioxidants would alter the redox balance with similar cellular outcomes. The involvement of oxidants in cytokine production is suggested by a study where H2O2 stimulated IL-8 release dose-dependently in human whole blood (DeForge et al., 1992). Significantly, oxidant scavengers inhibited the LPS-stimulated release of IL-8 (DeForge et al., 1992). The production of TNFα is also regulated by redox-dependent mechanisms since treatment of endotoxemic mice and dogs with the antioxidant N-acetylcysteine (NAC) reduces TNFα activity (Peristeris et al., 1992; Zang et al., 1994). Conversely, glutathione depletion with buthinone sulphoximine (BSO, an inhibitor of glutathione synthesis) exacerbates ROS induced-cell injury (Yang et al., 1995; Wakulich and Tepperman, 1997). This effect is not limited to glutathione-modulating agents because spin traps (which are chemical compounds that directly “trap” and thereby inactivate different classes of ROS) significantly reduce mortality when administrated in a murine endotoxic shock model 30 minutes before and 120 minutes after endotoxin (French et al., 1994).
b) Implications of ROS/NO Interaction for Preterm Birth
Another possibility for ROS to act as signal transducers is indirectly by modifying the bioavailability of another free radical, nitric oxide (NO) (Gryglewski et al, 1986). NO is an endogenously synthesized free radical produced by a variety of mammalian cells including neurons, smooth muscle cells, macrophages, neutrophils, platelets and others (reviewed by Nathan, 1992). Several groups report that a NO-cyclic guanosine monophosphate (cGMP) pathway exists in the rat (Yallampalli et al, 1992), rabbit (Sladek et al., 1993), guinea pig (Weiner et al., 1994) and human myometrium (Buhimschi et al., 1995). In all these species, the NO system is upregulated during pregnancy in either the myometrium or placenta (Yallampalli et al, 1992; Sladek et al.; 1993; Weiner et al., 1994; Buhimschi et al., 1995). This suggests to some that NO generation during gestation may contribute to the maintenance of uterine quiescence during pregnancy while its withdrawal prior to term may trigger parturition. A reversal of this scenario occurs in the cervix where the high NO output and inducible NO synthase expression occurs during rat labor suggesting a role for NO in the process of collagenolysis associated with cervical ripening (Buhimschi et al., 1996).
Several missing links emerge from the above sequence of events. How can increased NO produced in the uterus have such a dramatically different effect than NO produced in the cervix? Why do endotoxin-injected animals deliver prematurely despite a high nitric oxide production? (Buhimschi et al., 1996). One possible explanation is that the action of NO is modified by the oxidant/antioxidant balance as the coinciding spatial and temporal formation of high superoxide (O2.−) and NO amounts results in peroxynitrite (Huie et al., 1993), a powerful long-acting non-radical oxidant that oxidizes a number of biomolecules, including membrane phospholipids, sulfides, thiols, deoxyribose, as well as ascorbate and inhibits mitochondrial electron transport (Beckman and Crow, 1993). Peroxynitrite can also nitrate free or protein-associated tyrosine to generate nitrotyrosine (Ischiropoulos et al., 1992), which is considered as a marker for peroxynitrite action. The rate constant for the reaction generating peroxynitrite (i.e. the probability for the reaction to occur) is higher in systems that produce both NO and O2.− than those for the reactions between O2.− and SOD or NO and heme compounds (one of the clearance mechanisms of NO) (Pryor and Sqadrito, 1995). The possibility that under certain conditions the interaction between NO and ROS changes the effect of the free radical alone might explain the difference in the effects of a high NO output in the uterus (physiologically generated by non-inflammatory uterine cells during gestation) versus a high output of NO in the cervix (physiologically generated by inflammatory cells during ripening). Cervical softening may therefore be an example of a physiological spatially contained inflammatory reaction resulting in collagenolysis and tissue remodeling. It is well known that during the process of cervical softening the cervix becomes infiltrated with polymorphonuclear leukocytes (Junquiera et al., 1980). However, inflammatory cells produce large amounts of ROS (McCord et al., 1980) and of NO (MacMicking et al., 1997) and therefore peroxynitrite “provisionally”. In contrast the NO generating cells from the uterus during pregnancy produce only minimal O2.− and mostly by the endogenous xanthine oxidase activity (Telfer et al., 1997). In intrauterine infection or chorioamnionitis, an NO-ROS interaction can also occur in the uterus and by analogy, divert NO from its physiological role towards peroxynitrite, that in turn may contribute to the spread of a process ending in preterm delivery and poor fetal outcome.
c) Modulation of Matrix-Metalloprotease Activity by ROS
Matrix metalloproteases are a family of endopeptidases that collectively cleave most if not all the constituents of the extracellular matrix. Major members of this family are interstitial collagenase (MMP-1), 72-kDa type IV collagenase (MMP-2, 72-kDa gelatinase), stromelysin (MMP-3) and 92-kDa type IV collagenase (MMP-9, 92-kDa gelatinase). These enzymes are secreted into the intercellular compartment as proMMPs and require an activating agent to cleave apropeptide sequence and/or perturb their conformation. Autocatalytic processes, with further propeptide sequence cleavage, result in the fully active enzyme (reviewed by Brikedal Hansen et al., 1993).
Several MMPs (MMP-1, MMP-2, MMP-3, and MMP-9) are expressed in fetal membranes (Fortunato et al., 1997; Parry and Strauss, 1998). Increased MMP-9 activity in amniotic fluid (Athayde et al., 1998), human fetal membranes (Fortunato et al., 1997) and human plasma (Osmers et al., 1994) is reported during spontaneous labor at term. However, recent data suggest that preterm labor and preterm premature rupture of membranes are both associated with further elevated activity of MMP-9 in amniotic fluid (Athayde et al., 1998) and anmio-chorionic membranes (Fortunato et al, 1997). Modulation of matrix-metalloprotease activity by ROS may be relevant for preterm births that present initially with preterm premature rupture of membranes (PPROM). Significantly, these infants have a higher incidence of neonatal mortality and morbidity. In addition, the majority of infants with long term sequelae have documented PPROM before 24 weeks of gestation (Fanarroff et al., 1995).
MMP (particularly MMP-9) activation is a general feature of several inflammatory processes characterized by high cytokine output such as periodontal disease, rheumatoid arthritis and asthmatic airway inflammation (reviewed by Brikedal Hansen et al., 1993). Furthermore, the results of several in vitro experiments in culture conditions reveal a causative relationship between multiple cytokines (IL-1, IL-6, IL-8, TNFα; lipopolysaccharide: LPS) and both MMP-9 expression and activity (Fortunato et al., 1997; Esteve at al., 1998; Gottschall et al., 1995). In addition, modulation of the reduction/oxidation state of the environment alters MMP activity directly as well as the magnitude of the response induced by cytokines. Specifically, an increase in pro-oxidants or a decrease in antioxidants (i.e., altering the redox balance) increases MMP activity. Human heart fibroblasts are redox sensitive and under oxidative conditions are activated to concurrently express metalloproteases and TIMPs (Tyagi et al., 1996). Further, thiol (reduced glutathione and NAC) but not non-thiol reducing agents inhibit MMP activation and increase tissue inhibitors of MMPs expression in transformed cells. Incubation of cultured human vascular smooth muscle cells with a superoxide-generating mixture increases the amount of active MMPs, while NO donors have no noticeable effect (Rajagopalan et al., 1996). In cultured cartilage, antioxidants such as N-acetyl cysteine (NAC) and glutathione inhibit the chondrolytic activity of fibronectin fragments (Homandberg et al., 1996) that act through catabolic cytokine action mediated by IL-1, IL-6 and TNFα (Rathakrishnan et al., 1992). Endotoxin (LPS) activates MMP-9 in cultured microglia (Gottschall et al., 1995) and MMP-2 in cultured rat mesangial cells (Trachtman et al., 1996) and both IL-1 and TNFα reportedly operate at least partially through ROS (Rathakrishnan et al., 1992; Tiku et al., 1990),
It has also been shown that the fetal inflammatory response syndrome noted above is characterized by an outpouring of extracellular MMP-9 into the fetal circulation (Romero et al., 1998). High levels of MMP-9 are also found in the cerebral spinal fluid of patients with neurologic spastic diseases (Valenzuela et al., 1999) suggesting that MMP-9 may be a pathogenetical cause in the neural tissue remodeling described in infants with cerebral palsy.
Because of the known association between infection and preterm labor antibiotics are now proposed both as prophylaxis and treatment of preterm labor and delivery (Gibbs et al., 1997). Yet, antibiotics only prevent the release of new cytokines in addition to the microbial cytokine outpouring that occurs from microorganism killing. Said differently, antibiotics alone cannot reduce or inhibit the inflammatory process already underway.
Tocolytic drugs, which presently are the standard tool for the treatment of preterm labor despite their demonstrated inability to delay labor more than 48 hours (Higby et al., 1993), could actually have unintended adverse effects by prolonging the fetal exposure to an unfavorable environment and increase the probability of irreversible tissue damage and subsequently increased mortality or morbidity later in life.
Despite all the afore noted knowledge, no compounds have been put forth as therapeutic agents that address inflammation as a cause of preterm birth the fetal inflammatory process, PPROM, and adverse fetal outcomes.
d) Oxidative Stress: A Converging Point for Factors Causing Preterm Labor and Fetal Morbidity.
There are conditions other than infection and inflammation that stimulate oxidative stress during pregnancy. Cocaine, smoking and alcohol consumption are each well known for this property, although the nature of, and the biochemical pathways by which free radicals are generated in vivo differ. We believe that therapeutic agents as NAC or other antioxidants and ROS scavengers are beneficial in such instances.
Cocaine use is associated with intrauterine growth restriction, stillbirth, placental abruption and congenital malformations (limb reductions). The incidence of preterm labor ranges in cocaine users from 20 to 50% (Little et al., 1989; Feldman et al., 1992). The placental abruptions and cerebral infarcts found in neonates exposed to cocaine in utero are attributed to the vasoactive effect of cocaine, which inhibits the uptake of catecholamines and serotonin by nerve endings. The net result is vasoconstriction and an ischemic-reperfusion injury. (Chasnoff et al., 1985). Animal studies confirm that the teratogenic effect of cocaine is secondary to vasoconstriction and local hemorrhage. (Webster and Brown-Woodman, 1990). In addition, cocaine increases the placental thromboxane to prostacyclin ratio. (Monga et al., 1994). As noted previously, the first oxygen free radical generated by an ischemic-reperfusion injury is O2− produced via the activation of xanthine oxidase in the oxygen-deprived tissue. This is followed by mitochondrial leakage and lastly inflammatory cell recruitment in the area of necrosis. Alternatively, cocaine produces excess free radical metabolites during its metabolism in the hepatic microsomes, a possibility that could explain the hepatotoxicity of cocaine (Boelsteri et al., 1992). Cocaine also affects the fetus through maternal injury since vascular disruptions and free radical injury does not occur in embryos co-cultured with cocaine, and the maternal administration of a specific antioxidant inhibits lipid peroxidation by cocaine (Zimmerman et al., 1994). Rat fetuses exposed to cocaine display accelerated lung maturation unparalleled by an induction of antioxidant enzymes (Sosenko, 1993). These studies suggest that cocaine use results in oxidative stress with profound fetal consequences. In addition to the genesis of oxidative stress, acute cocaine exposure increases myometrial contractile activity (Monga et al., 1993a; Monga et al 1993b) by increasing intracellular calcium (Formin et al., 1999) yet another mechanism that may contribute to preterm delivery.
Ethanol consumption during pregnancy is associated with preterm labor in addition to its well-publicized fetotoxic effects. The fetal toxicity of ethanol is polymorphic. The described fetal alcohol syndrome includes pre- and postnatal growth disturbance, mental retardation, heart defects, limb defects and a characteristic facial anomaly. (Jones et al., 1973). It is the most commonly identified cause of mental retardation. A significantly increased risk of white matter brain damage is also reported (Holzman, 1995). The underlying mechanism remains largely unknown. In susceptible strains of inbred mice, ethanol produces preterm labor by initiating a cascade of events similar to endotoxin (Salo et al., 1996; Cook et al., 2000). The biochemical alterations induced by ethanol include delayed cell replication, altered membrane fluidity and transport mechanisms that have been linked to oxidative stress-induced membrane damage (Henderson et al., 1999; Kourie, 1998).
There is abundant literature on ethanol-induced free radical formation. These studies confirm that hepatic macrophage NADPH oxidase is the primordial source of O2− radicals and H2O2, and that their levels can be reduced by antioxidant treatment (Nanasumrit et al., 2000). These reactive oxygen intermediates subsequently lead to the formation of 1-hydroxyethyl radicals. Knockout mice lacking NADPH oxidase are resistant to ethanol-induced 1-hydroxyethyl radicals formation and hepatic injury (Kono et al., 2000).
Cigarette smoking during pregnancy is another risk factor for preterm delivery. A recent meta-analysis restricted to prospective studies revealed a 1.27 pooled odds ratio (95% confidence interval, 1.21-1.33) with a dose response relationship at low to moderate levels of smoking (Shah and Bracken, 2000). Another recent study suggested that multiparous women have an even higher risk (Kolas et al., 2000).
One of the mechanisms by which smoking induces DNA damage and lung cancer is via HO. radical-mediated mutagenesis (Pourcelot et al., 1999). Of particular interest is the observation that hypoxia produced by smoking triggers the up-regulation of a circulating antioxidant (reduced glutathione) and vasodilator mechanisms. For example, chronic and acute smoking results in a high production of cyclic guanosine monophosphate (cGMP: a vasodilating second messenger of NO) in both urine (Markovitz et al., 1997) and plasma (Dupuy et al., 1995). The level of reduced glutathione in erythrocytes is 35% higher in pregnant women who smoked compared to non-smokers (Laskowska-Klita et al., 1999). Although maternal smoking is associated with higher risk of preterm delivery and a net detrimental effect on pregnancy outcome, these compensatory mechanisms may contribute to the observation that smokers have a lower risk of preeclampsia (Zhang et al., 1999) a disorder where vasoconstriction, hypoxia and possibly decreased endothelial cGMP production are part of the pathophysiologic process.
Sickle cell anemia, thalassemia and glucose-6-phosphate-dehydrogenase deficiency are all hereditary anemic disorders with higher potential for oxidative damage due to chronic redox imbalance in red cells (lower reduced GSH) that often results in clinical manifestation of mild to serve hemolysis in patients with these disorders (Chan et al., 1999). Women with these hereditary anemic disorders have a higher risk of preterm delivery and poor neonatal outcome (Seoud et al., 1994). It has been observed that NAC has the ability to cause a significant diminishment of sickle cell formation in vitro while other antioxidants had no effect (Xunda et al., 1998; Shartava et al., 1999).
Preeclampsia, a syndrome unique to primates and defined by the triad of hypertension, proteinuria and pathological edema during pregnancy had been associated with oxidative stress affecting maternal endothelium. In support of this conclusion, a recent, randomized trial of women at risk for preeclampsia concluded that supplementation with vitamins E and C was effective in preventing occurrence of early markers or symptoms of preeclampsia. The present inventors propose that NAC alone or in combination with other antioxidants to will increase the therapeutic efficacy in preeclampsia.
The present inventors and others have previously reported the chronic competitive inhibition of NO synthesis with L-arginine analogues (NG-ritro-L-arginine methyl ester: L-NAME) can cause hypertension, proteinuria and fetal growth restriction in rats without affecting gestational length if pregnant (Baylis et al., 1992; Yallampalli and Garfield 1993; Molnar et al., 1994). Glomerular damage and histopathological changes in the placental bed similar to human preeclampsia are suggested (Osawa, 1996). The increased blood pressure and fetal growth restriction are reversed by simultaneous infusion of L-arginine but not D-arginine (not a substrate for NOS) (Buhimschi et al., 1995; Liao et al., 1996) The fetuses from L-NAME treated rats frequently exhibit distal limb necrosis (Dicket et al., 1994)/A recent study suggests that chronic NO inhibition promotes a state of oxidative stress with HO-mediated DNA damage (Tsukahara et al., 2000). Recent findings from our group reveal that some colonies of rats are refractory to NO inhibition and the symptoms of preeclampsia do not occur despite continuous L-NAME infusion throughout pregnancy (Buhimschi et al., in press). This strongly suggests that the extent of clinical manifestations in preeclampsia may reflect the interaction of a multitude of antioxidant and vasodilatory mechanisms that can compensate for one another to some extent.